Volume 22, Issue 8, Pages 720-726 (April 2012) Ribonucleotide Reductase Activity Is Coupled to DNA Synthesis via Proliferating Cell Nuclear Antigen  Israel Salguero, Estrella Guarino, Marianne E.A. Shepherd, Tom D. Deegan, Courtney G. Havens, Stuart A. MacNeill, Johannes C. Walter, Stephen E. Kearsey  Current Biology  Volume 22, Issue 8, Pages 720-726 (April 2012) DOI: 10.1016/j.cub.2012.02.070 Copyright © 2012 Elsevier Ltd Terms and Conditions

Current Biology 2012 22, 720-726DOI: (10.1016/j.cub.2012.02.070) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 Increased Expression of cdt2 Is Necessary but Not Sufficient to Induce Spd1 Proteolysis (A) Cdt2-TAP levels in exponentially growing (log) or mitotically arrested wild-type cells (2710) and exponentially growing yox1Δ mutant cells (2698). (B) Western blot analysis showing Spd1-TAP levels after methyl methanesulfonate (MMS) addition in a wild-type strain (1766) and in a rad3Δ (2644) mutant (upper panels), Spd1-TAP levels after MMS treatment of yox1Δ (2711) and rad3Δ yox1Δ (2713) cells (middle panels), and Spd1-TAP levels after MMS treatment of mitotically arrested cells that were either wild-type for checkpoint function (2678) or DNA damage checkpoint defective rad3Δ (2677) mutant (lower panels). (C) Spd1-TAP levels in exponentially growing (log) or mitotically arrested wild-type cells (2678). Tubulin is shown as a loading control. Current Biology 2012 22, 720-726DOI: (10.1016/j.cub.2012.02.070) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 Chromatin-Bound PCNA Is Required for Spd1 Proteolysis (A) Spd1-TAP levels in a wild-type strain (1766) and in an rfc1-44 thermosensitive mutant (2072), treated with hydroxyurea (HU) or MMS at the restrictive temperature (37°C). (B) Western blot analysis showing Spd1-TAP levels after HU or MMS addition in a wild-type strain (1766) and a pcn1D122A mutant (2649). (C) Spd1-TAP levels after MMS treatment of pcn1D122A yox1Δ (2755) cells and mitotically arrested (nda3) pcn1D122A (2664) cells. Tubulin is shown as a loading control. See also Figure S1. Current Biology 2012 22, 720-726DOI: (10.1016/j.cub.2012.02.070) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 PCNAD122A-Induced Defects Are Suppressed by spd1 Deletion (A) pat1 strains also containing pcn1D122A (2912), pcn1D122A spd1Δ (2915), or spd1Δ (2930) mutations were arrested in G1 by nitrogen starvation and then released from the block at 34°C to inactivate Pat1 and induce meiosis. The progress of premeiotic S phase was followed by flow cytometry. (B) Upper panels: images of exponentially growing cells from wild-type (137), spd1Δ (2671), rad3Δ (1811), pcn1D122A (2738), pcn1D122A spd1Δ (2747), and pcn1D122A spd1Δ rad3Δ (2842) cultures. Lower panel: average cell length ± SD (150 cells were measured for each strain). (C) Viability of wild-type (137), pcn1D122A (2738), spd1Δ (2671), rad3ts (2839), pcn1D122A rad3ts (2887), pcn1D122A spd1Δ rad3ts (2888), spd1Δ rad3ts (2889), and pcn1D122A spd1Δ (2747) strains on YE3S at 26°C and 37°C analyzed by spot tests. (D) Rate of minichromosome loss in wild-type (2836), pcn1D122A (2898), spd1Δ (2885), and pcn1D122A spd1Δ (2900) strains. See also Figure S2. Current Biology 2012 22, 720-726DOI: (10.1016/j.cub.2012.02.070) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 Spd1 and PCNA Interact In Vivo, and a PIP Box in Spd1 Is Important for This Interaction (A) Identification of a sequence (orange bar and box) in S. pombe Spd1 (gray bar) that partially matches the PIP degron consensus sequence. Other sequences shown are (bottom to top) corresponding region of S. japonicus Spd1 (SjSpd1), PIP degrons in S. pombe Cdt1 (SpCdt1_301 and SpCdt1_28 [18]), PIP degron and PIP box consensus sequences [25], and the mutated sequence of Spd1MPIP. Green and blue bars at bottom show the Spd11–43 and Spd144–124 deletion mutants. The cyan bar labeled “R1” represents the location of sequence similarity with a region of Sm11 known to interact with the R1 subunit of RNR [11]. (B) Western blot analysis of Spd1-YFP-NLS levels after HU addition in a wild-type strain (2672), the Spd11–43 mutant (2612), the Spd144–124 mutant (2744), and the Spd1MPIP mutant (2673). Tubulin is shown as a loading control. (C) Bimolecular fluorescence complementation (BiFC) of exponentially growing live cells expressing VN173-pcn1 and spd1-VC155 in a wild-type (2536) and in a cdt2Δ (2546) background. (D) Comparison of the intensity of BiFC in exponentially growing live cells. A wild-type (2752) and an spd11–43 (2750) strain (left panels), a wild-type (2752) and an spd144–124 (2751) strain (middle panels), and a wild-type (2752) and an spd1MPIP (2748) strain (right panels) were compared. Phase (blue) and BiFC signal (green) channels are shown merged (upper panels). In all combinations, cells carrying the mutation in Spd1 were stained with MitoTracker Red to allow direct comparison between the two strains (lower panels). All strains were in a cdt2Δ background to stabilize Spd1; comparison of Spd1 levels in these strains is shown in Figure S3B. (E) Model for RNR activity control by PCNA. Left panel represents an unperturbed cell in G1, G2, or M phase that is not subject to DNA damage. Spd1 shuttles between nucleus and cytoplasm, interacting with and inhibiting RNR in the cytoplasm and possibly interacting with free PCNA in the nucleus. CRL4Cdt2 does not ubiquitylate nuclear Spd1, as PCNA is not DNA bound. Right panel represents the situation when S phase starts or DNA synthesis associated with DNA damage repair is occurring. Spd1 shuttles from cytoplasm to nucleus, but in the nucleus, following interaction with chromatin-bound PCNA, it is ubiquitylated and proteolyzed. The net reduction in Spd1 levels leads to RNR activation. Current Biology 2012 22, 720-726DOI: (10.1016/j.cub.2012.02.070) Copyright © 2012 Elsevier Ltd Terms and Conditions